Method for identifying active substances

ABSTRACT

The invention relates to a method and a device for identifying active substances in order to determine the formation of complexes between reactants. According to the inventive method, at least two reactants are mixed and made to react to form a complex. The infrared spectrum of the individual reactants which have not yet been reacted in the mixture is measured during a first time interval and at least a second infrared measurement is conducted to measure the complex formed by the reactants during a second interval. The difference between the two spectra measured during different moments is then determined. The reactants whose differential spectrum exhibits a band structure are selected as active substances.

BACKGROUND OF THE INVENTION

The invention relates to a method for identifying active substances anda device for executing the method.

In “Fourier-Transform Infrared Spectroscopic Studies on Avidin SecondaryStructure and Complexation with Biotin and Biotin-Lipid Assemblies,”Biophysical Journal Vol. 71 (1996), pp. 840-847, M. J. Swamy, T.Heimburg and D. Marsh describe efforts to explain the structure ofcomplexes of the protein avidin with biotin and biotin-lipid throughFourier-transform infrared spectroscopy (FTIR). In this work, FTIRspectra of the avidin are first recorded in heavy water (D₂O). Then,avidin with biotin or biotin-lipid is mixed with buffered D₂O as asolvent and stored for several hours at room temperature, whichapparently should produce the highest yield of the resulting avidincomplex. The FTIR spectra of the complex are recorded again.Differential spectra are formed from the spectra of the avidin and thespectra of the avidin complex. Because this work only focuses on thestructure of the avidin complexes, no time-dependent spectra arerecorded. In all instances, the described FTIR spectra reflect states ofequilibrium. The vibrational spectra of bonded deuterium isrecorded—shifted due to the higher mass—as opposed to the vibrationalspectra of bonded, normal hydrogen.

Consequently, a comparatively thick cuvette (50 μm) can be used. Thequestion of whether a chemical compound, such as a protein, may form ata coordination point with a specific ligand is of no consequence in thiswork, because the fact that a complex of avidin with biotin forms wasalready known.

FTIR studies for determining structures of protein complexes are alsodescribed by M. Gonzales, et al. in “Interaction of Biotin withStreptavidin,” The Journal of Biological Chemistry, Vol. 272, No. 17(1997), 11288-11294. The spectra were recorded with an H₂O buffer aswell as a D₂O buffer; in the case of H₂O, the layer thickness was 6 μm,and 50 μm in the case of D₂O. The goal of the study was to ascertain thethermal stability of biotin and the biotin-streptavidin complex. Thethermal denaturing was represented in chronologically consecutivespectra. In contrast, the formation of the complexes was notinvestigated with spectrometry.

In “Redox-linked conformational changes in proteins detected by acombination of infrared spectroscopy and proteinelectrochemistry—Evaluation of the technique with cytochrome c,” Eur. J.Biochem. 187, 565-572 (1990), D. Moss, E. Nabedryk, J. Breton and W.Mantele report on an electrochemical reduction and a subsequentre-oxidation of the protein cytochrome c. Cytochrome c is provided in alayer thickness of 10 to 15 μm to preclude the IR absorption of water inthe medium infrared range. The reduction and subsequent re-oxidationwere proven with the aid of FTIR spectroscopy. Differential spectra ofthe reduced and re-oxidized state are shown. Because the cuvette onlycontained cytochrome c, no definitive statements could be made about theformation of protein complexes.

The publication by A. J. White, K. Drabble and C. W. Wharton: “Astopped-flow apparatus for infrared spectroscopy of aqueous solutions,”Biochem. J. (1995) 306, 843-849 describes an apparatus for executing theso-called “stopped-flow” method, in which the reagents are sprayed intoa cuvette with sprayers, and mixed. According to the authors, HPLCvalves have proven unsuitable due to the necessary high pressure and thehigh viscosity of peptides. This apparatus was used to record FTIRspectra in the temporal range of 6.25 seconds to 966 seconds after themixing of 12C═O- and 13C═O-cinnamoyl chymotrypsin with a deacylatingagent in a D₂O buffer; the optical layer thickness was 50 μm.Differential spectra were formed from the spectra of 12C═O- and13C═O-cinnamoyl chymotrypsin. The “Conclusions” include the statementthat it is not possible to create a “stopped-flow” IR transmissioncuvette that permits the use of (non-deuterated) water, because theheavy absorption of water at 1640 cm⁻ requires a layer thickness of 5 μm(the writings incorrectly state ‘5 mm’).

Q. H. Gibson and L. Milnes provide a detailed description of the“stopped-flow” method in “Apparatus for Rapid and SensitiveSpectrophotometry,” Biochem. J. (1964) 91, 161-171.

The large dead volume of the apparatuses due to the use of sprayers is ageneral drawback of the “stopped-flow” methods. Mass-screening methods,therefore, cannot be implemented with such apparatuses, notably becausethe microtitration plate provided with 96 depressions of 400 μl each isthe standard model for automated methods; refer to J. R. Broach and J.Thorner: “High-throughput screening for drug discovery,” Nature Vol. 384Supp. Nov. 7, 1996, which offers an overview of mass-screening methods.With regard to ascertaining the bondability of a ligand to the receptorof a peptide, Broach and Thorner cite a method in which Eu²⁺ at theligand and allophycocyanin at the receptor are covalently bonded.Through the formation of a receptor-ligand complex, Eu²⁺ closelyapproaches allophycocyanin, resulting in an energy transfer that can bedetected as a fluorescence signal.

SUMMARY OF THE INVENTION

It is an object of the invention to develop a method for identifyingactive substances that permits the low-cost detection of the formationof complexes between reactants in the smallest-possible volume, flexiblyand quickly and with reproducible results. The method is also intendedto have the capability of being automated. It is a further object toprovide a device for executing the method.

The above and further objects are accomplished according to theinvention by the provision of a method comprising: mixing at least tworeactants that form a reactant complex; recording an IR spectrum ofindividual reactants that have not yet been converted in the mixture ata first time; recording at least one further IR spectrum at a secondtime for detecting the reactant complex; forming a differential spectrumfor the two IR spectra recorded at different times; and selecting thereactants whose differential spectrum has a band structure as activesubstances.

According to the invention, the active substances are identified throughthe investigation of the bondability of a reactant, such as a ligand, toat least one further reactant, such as a protein. The reactants producea mixture from which an IR or FTIR spectrum is recorded at least at twodifferent times. The ligand measurement can be effected in an aqueoussolution, in which case the conventional use of deuterated solutions,such as deuterated water or a deuterated buffer, is not absolutelynecessary. Depending on the viscosity and the physical-chemicalproperties of the reactants, the use of a different or further solventmay be indicated; a deuterated buffer or deuterated solvent can beomitted.

The mixture can be produced in accordance with the cited state of thetechnology. The use of high-pressure pumps (up to about 400 bar) and theloop valves known from HPLC technology is preferred, however.

The mixture should preferably be applied in a layer thickness of 1 to 25μm, especially 8 to 15 μm. The mixture is advantageously produced on theway to and/or in an IR cuvette with a corresponding optical thickness.

Usually, one endeavors to produce a complete mixture from the organiccompound and the reagent. Because most of the reactions of organiccompounds take place slowly, the time required to produce an optimummixture and record the first IR or FTIR spectrum is typicallysufficiently short. If, however, the speed of reactions between thereactants is high, it may be advisable to record the first IR or FTIRspectrum with an incomplete mixture to prevent a substantial reactionconversion at this time.

In the recording of the first IR or FTIR spectrum, the reactants muststill be at least partially unconverted, so the formation of the complexcan be detected in the second or further IR or FTIR spectrum. Ideally,the reaction of the reactants should not have begun after the mixing.This is impossible for very rapid reactions due to the mixing prior toand/or on the way to the IR cuvette, so a portion of the reactantsshould have already reacted with one another. In the method of theinvention, a partial reaction of the reactants is not problematic,provided that sufficient quantities of the reactants can react with oneanother and a measurement signal can be obtained. If the recording ofthe first IR or FTIR spectrum reveals that an inadequate portion of thereactants is present in unconverted form, only spectra no longerpossessing sufficient differences can be recorded. The differentialspectra in this case exhibit a zero line.

Preferably, a first FTIR spectrum is recorded immediately after themixture is produced (time t₀). “Immediately” means that the spectrum isrecorded as quickly as technically possible. Because the reaction speedis the highest immediately after the reactants have come into contactwith one another, it is crucial for the rapidity and precision of themethod that the reaction speed still be sufficiently high during therecording of the first spectrum, and that the primary portion of thereactants not react until afterward. Above all, in slower reactions, itis possible to wait for some time before recording the first spectrum,as long as the reaction speed of the reactants is sufficiently high tobe measured. The first spectrum, however, is advantageously recordedwithin one to 1000 milliseconds after the reactants have been mixed.

In the method of the invention, differential spectra of two spectra thatare recorded at arbitrary times are formed for identifying the activesubstance. The differential spectrum is preferably formed with thespectrum that is recorded immediately after the mixing of the reactants(measurement time t₀). A spectrum that has a large temporal spacing fromthis spectrum is preferably selected as the second spectrum. If morethan two reactants are used in the measurement, the reaction ispreferably started by the addition of the reactant that leads to thecomplex formation being investigated.

Substances whose pharmaceutical or phytopharmacy effect is presumed, andwhich are supposed to be investigated more closely, are used asreactants. Reactants can include, for example, potential medicines,potential herbicides, fungicides or insecticides that are capable offorming complexes.

The active substances in the method of the invention should preferablyencompass those substances that exhibit a physiological effect in theplant, animal or human body, e.g., hormones, vitamins, enzymes,pharmaceuticals or pesticides. Active substances are reactants such asproteins, e.g., enzymes such as ECE or ACE, receptors, such as glutamatereceptors, antibodies, protein inhibitors such as PAI, mediators, e.g.,interferons such as gamma interferon, interleukins such as interleukin-2or interleukin-6, transcription factors such as Sp1, regulator proteins,translocators or chaperones.

Low-molecular substances having an average molecular weight in apreferred molecular-weight range of 100 to 10,000 Daltons (=d),especially in a range of 100 to 1000 d, are also mentioned here.Low-molecular substances encompass organic chemical compounds that maycontain, for example, substituted aliphatic or aromatic heterocyclene,aromatics, saturated or unsaturated aliphatics, amines, ketones,thioketones, alcohols, thiols, esters, amides, ethers, thioethers,nitriles, isonitriles, aldehydes or their derivatives.

Active substances that, by way of the release of a ligand which, as areactant, ultimately forms a complex with the further reactant(s), canalso be detected with the method of the invention.

In the method of the invention, the identification of complex formationsbetween proteins is less preferable, because proteins cannot beadministered orally, for example as active substances, and frequentlycause allergic reactions. In the method of the invention, the complexformation between proteins, DNA or RNA and low-molecular substances ispreferably investigated. In the method of the invention, at least one ofthe reactants can be a protein or a DNA; at least one further reactantshould be a low-molecular substance. Interactions between long- andshort-chain DNA or RNA can also be detected.

Primarily in the complex formation of and with proteins, but also inmany other organic compounds, the medium IR range between 2500 and12,500 nm is preferably used.

After a waiting period, a second IR or FTIR spectrum is recorded (timetn). The length of the waiting period (=x) depends on the reaction speedof the reaction partners. The length of the waiting period is between 1ms and one day, preferably between 10 ms and 120 min, especially between10 ms and 10 min. If one reactant is a protein, a waiting period in therange of aa5 to 30 s, e.g., 20 s, is suitable. Much shorter waitingperiods, for example in the range of 10 to 100 ms, are required forinvestigating the avidin/biotin complex, for example. Waiting periods inthe minute range should be implemented for the reaction of someantibodies and in the hybridization of DNA.

The reaction conversion that has taken place to this point is documentedin the second IR or FTIR spectrum. This reaction conversion can berepresented through the formation of a differential spectrum between thefirst spectrum, for example at t₀, and the second spectrum at t_(n). Thedifferential spectrum is formed according to ΔA_(v)=¹⁰log(I_(1v)/I_(2v)), where I_(1v) and I_(2v) are the measured intensities atthe frequency v in the first and second spectrum, respectively.

If the differential spectrum essentially comprises a straight line, noreaction has occurred, because in this case the concentration of thereaction partners does not change. A bonding of the reactants istherefore manifested in a differential spectrum having a band structure.

In the method of the invention, complexes include all covalently ornon-covalently bonded reactants or their components. Non-covalent bondsinclude bonds formed by van-der-Waals forces, a hydrogen-bridge bond orionic bonds. According to the invention, non-covalently bonded reactantsor their components are preferred.

A significant merit of the method according to the invention is that themethod can be automated, so the reactant complexes can be formedquickly, either consecutively or in parallel. So-called mass-screeningmethods are particularly significant for the development of newmedications. In this process, a protein that is associated with themanifestation of an illness, for example, is identified. The object isto find a suitable medication that inhibits this so-called targetprotein. The target protein can be obtained from the correspondingbiological material in large quantities and with high purity. Numerousreagencies whose pharmaceutical effectiveness is presumed are tested todetermine whether they possess the desired inhibitory effect. The numberof reagencies to be tested is generally very large, even a five-, six-or seven-digit number, so the use of a fast, automatic screening methodis of critical economic significance.

This object can be accomplished by the method according to theinvention. The high scanning speeds of modern FTIR spectrometers and theshort time required for executing the method establish the prerequisitesfor testing a large number of reagencies. A further notable advantage ofthe method is that the conventional, standard microtitration plates canbe used without problems. Because the trend is toward even smallermicrotitration plates having 384 or even 864 holes, which have a lowervolume of about 100 μl or about 50 μl, the small sample quantityrequired for the method and device of the invention is especiallysignificant. Further important advantages of the method, and the reasonsthat the method is suitable for mass-screening methods, are that nodeuterated water need be used, and the device can be used without anystructural modifications for numerous studies.

BRIEF DESCRIPTION OF THE DRAWINGS

An embodiment of the device for executing the method of the invention isdescribed in conjunction with the figures, which show in:

FIG. 1 the embodiment of the device in a first operating state;

FIG. 2 the embodiment of the device in a second operating state; and

FIG. 3 the embodiment of the device in a third operating state.

DETAILED DESCRIPTION OF THE INVENTION

FIG. 1 shows the device in a first operating state, in which the valves3 and 4 are filled with a test substance or a target protein. The testsubstances are placed on an xyz positioning table in a standardmicrotitration plate 11, and the target protein is placed in a reservoir9. The valves 3 and 4 are turning valves (by the Valco company) knownfrom HPLC technology; in the illustrated position, they are switchedsuch that the two low-pressure pumps 6 and 7 can fill them with a testsubstance from the standard microtitration plate 11 or the targetprotein from the reservoir 9. In both cases, any excess is conductedinto the sewer system (not shown).

In the operating state illustrated in FIG. 2, the valves 3 and 4 arereversed. The test substance and the target protein are mixed with theaid of the high-pressure pump 8, which is connected to a reservoir 10for distilled water, and transported into the IR cuvette 2 in the FTIRspectral photometer 1.

FIG. 3 illustrates the operating state in which the first FTIR spectrum(t₀) and, after a waiting period, the second FTIR spectrum (t_(n)), arerecorded. The valve 5 is reversed here, so the test substance and thetarget protein are isolated in a closed line system.

After the spectra are recorded, the measurement cell is rinsed withdistilled water before the cycle begins again.

The device is essentially constructed from HPLC components. Thehigh-pressure pump 8 is an HPLC pump (by the Alltech company), which iswell-suited for continuous high-pressure operation. All fluid lines andconnections comprise HPLC components that can be loaded up to 400 bar.To reduce wear and tear on the HPLC pump, only distilled water ispumped. The selected arrangement of the HPLC turning valves 3, 4 and 5assures the additional advantages of efficient cleaning of the system,and a reduced consumption of sample material, which are significant formass screening. With the aid of the turning valve 5, the high-pressurepump 8 diverts, rather than stops, the flow of distilled water from thereservoir 10, so the flow through the IR cuvette can be stopped. Thisprocess reduces wear and tear on the pump, resulting in a fasterresponse time. Moreover, this short-circuits the intake and dischargelines of the IR cuvette, which quickly compensates the overpressure andeliminates changes in layer thickness due to bulging of the IR cuvette.

The device illustrated in the figures can be used to achieve throughputspeeds of up to 40 ml/min, which correspond to an exchange of thecontents of the IR cuvette within 15 ms. A pressure of up to about 150bar is recorded. The valve 5 diverts the fluid flow in 20 ms. Itsuffices to use 100 μl each of the target protein and the test substancefor recording the FTIR spectra; this amount can be reduced further by afactor of 3 to 5 through the optimization of the sample requirement.

What is claimed is:
 1. A method for identifying active substances,comprising: mixing at least two reactants that form a reactant complex;recording an IR spectrum of individual reactants that have not yet beenconverted in the mixture at a first time; recording at least one furtherIR spectrum at a second time for detecting the reactant complex; forminga differential spectrum for the two IR spectra recorded at differenttimes; and selecting the reactants whose differential spectrum has aband structure as active substances.
 2. The method according to claim 1,further comprising: selecting a time immediately following the mixing ofthe reactants as the first time.
 3. The method according to claim 1,wherein at least one of the reactants is a low-molecular compound. 4.The method according to claim 1, wherein at least one of the reactantsis a protein.
 5. The method according to claim 1, wherein at least oneof the reactants is a DNA molecule.
 6. The method according to claim 1,wherein there is a waiting period in a range of one millisecond to oneday between the first and second times.
 7. The method according to claim1, including placing the reactants in a microtitration plate andexecuting the method successively or in parallel with a plurality ofreactants.
 8. The method according to claim 1, including measuring theIR spectra in a layer thickness of 1 to 25 μm.